PROJECT ABSTRACT Interactions between sperm and the female reproductive tract (FRT) are critical to fertility. A new paradigm is emerging, based on reproductive studies across a diversity of animal taxa, that emphasizes the contribution of post-copulatory male-by-female interactions to reproductive outcomes. Whereas it is evident that these interactions influence the physical aspects of sperm migration through the FRT, they also include, for example, cell-to-cell signals between sperm and the epithelium that are critical for sperm progression to the oviduct, stimulate ovarian secretions and induce sperm modifications necessary for fertilization competency. However, little is known about the molecular nature of these interactions, how sperm are modified or how these changes influence sperm motility, viability and fertilization competence. Studies proposed here will substantively advance our understanding of the in vivo molecular fate of sperm using the model species Drosophila melanogaster. First, we will use sex-specific metabolic labeling and mass spectrometry to identify novel male and female protein interactions with sperm in the FRT and assess the specificity of these interactions. Second, a refined quantitative landscape of sperm proteome modifications, including post-translational modification, following insemination and after protracted storage, will be achieved using isobaric labeling proteomic techniques. Third, the powerful genetic toolkit available in Drosophila will be used to conduct RNAi knockdowns of novel male sperm-associated proteins, and combined with use of fluorescent-tagged sperm and methods for direct visualization of real-time sperm function within the FRT, to directly assess their impact on sperm motility, storage, survival and fertilization competency, in addition to an array of female traits relevant to fertility. The exhaustive and complementary proteomic datasets generated across various stages of the postcopulatory ?life history? of sperm will substantially expand our understanding of sperm maturation in the FRT and serve as a platform for targeted studies of genes governing sperm-by-female interactions critical to fertility.